Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in diseases such as leukemia. Here, we describe E/L repli-seq, an extension of our repli-chip protocol. E/L repli-seq is a rapid, robust and relatively inexpensive protocol to analyze RT by next-generation sequencing (NGS), allowing genome-wide assessment of how cellular processes are linked to RT. Briefly, cells are pulse labeled with BrdU and early and late S phase fractions are sorted by flow cytometry. Labeled nascent DNA is immunoprecipitated from both fractions and sequenced. Data processing leads to a single bedGraph file containing the ratio of nascent DNA from early versus late S phase fractions. The results are comparable to repli-chip, with the additional benefits of genome-wide sequence information and an increased dynamic range. We also provide computational pipelines for downstream analyses, for parsing phased genomes using single nucleotide polymorphisms (SNP) to analyze RT allelic asynchrony, and for direct comparison to repli-chip data. This protocol can be performed in up to three days prior to sequencing, and requires basic cellular and molecular biology skills and a basic understanding of Unix and R.

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Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early and late replicating regions differ in terms of mutation rates, transcriptional activity, chromatin marks and sub-nuclear position. Moreover, RT is regulated during development and is altered in disease. Exploring mechanisms linking RT to other cellular processes in normal and diseased cells will be facilitated by rapid and robust methods with which to measure RT genome wide. Here, we describe a rapid, robust and relatively inexpensive protocol to analyze genome-wide RT by next-generation sequencing (NGS). This protocol yields highly reproducible results across laboratories and platforms. We also provide computational pipelines for analysis, parsing phased genomes using single nucleotide polymorphisms (SNP) for analyzing RT allelic asynchrony, and for direct comparison to Repli-chip data obtained by analyzing nascent DNA by microarrays. INTRODUCTIONDNA replication occurs during S phase of the cell cycle. In human cells, this process lasts around 8 hours 1 . Different regions of the genome replicate at different times during S phase, following a defined replication timing (RT) program 2,3,4,5,6,7,8 . RT is highly conserved between mouse and human 4,5,6,9 . Interestingly, in mammalian cells, almost 50% of the genome switches RT upon cell differentiation 2,3,4,5,6,8 . RT is linked to transcription, although the causal links between these two processes are not clearly understood 10,11,5,12,8,13 . Moreover, RT is closely associated with 3D nuclear architecture 6,9 as measured by chromatin conformation capture methods such as Hi-C 14 . Early and late replicated regions correlate with the A and B compartments identified by Hi-C analysis 5,6,14,15 , while domains of coordinately regulated RT (replication domains; RDs) align with topologically associating domains (TADs) 15 . Accurate methods to analyze RT are essential to explore the links between all these processes.Genome-wide RT analysis methods are based on the quantification of replicated genomic regions at different times during S phase. Multiple techniques have been developed to assess RT. One of the major applications, repli-chip, uses BrdU pulse labeling of nascent DNA, Fluorescence Activated Cell Sorting (FACS) to separate cells into different times during S phase 16,17 , and BrdU immunoprecipitation to isolate newly synthesized DNA at different time points of the S phase 2,18 . This newly synthesized DNA is then quantified by microarray hybridization. Subsequently, Hansen et. al. sequenced the newly synthesized DNA produced from BrdU labeling and FACS sorting to coin the term Repli-seq 4 . In either case, the number of S phase fractions can be varied in this protocol, from 2 fractions (early vs. late or E/L), which produces a simple ratio of enrichment in early vs. late S phase, to multiple fractions (to date up to 8) of S phase 4,16,17,19 . Both 2 and 6 fraction Repli-chip vs. Repli-seq give highly similar profiles after smoothin...

This study was designed to investigate the potential effects and underlying mechanism of adipose tissue-derived mesenchymal stem cells (MSCs) on allergic inflammation compared to Montelukast as an antileukotriene drug in a rat model of allergic rhinitis (AR). The effect of MSCs was evaluated in albino rats that were randomly divided into four (control, AR, AR + Montelukast, and AR + MSCs) groups. Rats of AR group were sensitized by ovalbumin (OVA) and then challenged with daily nasal drops of OVA diluted in sterile physiological saline (50 μL/nostril, 100 mg/mL, 10% OVA) from day 15 to day 21 of treatment with/without Montelukast (1 h before each challenge) or MSCs I/P injection (1 × 106 MCSs; weekly for three constitutive weeks). Both Montelukast and MSCs treatment started from day 15 of the experiment. At the end of the 5th week, blood samples were collected from all rats for immunological assays, histological, and molecular biology examinations. Both oral Montelukast and intraperitoneal injection of MSCs significantly reduced allergic symptoms and OVA-specific immunoglobulin E (IgE), IgG1, IgG2a and histamine as well as increasing prostaglandin E2 (PGE2). Further analysis revealed that induction of nasal innate cytokines, such as interleukin (IL)-4 and TNF-α; and chemokines, such as CCL11 and vascular cell adhesion molecule-1 (VCAM-1), were suppressed; and transforming growth factor-β (TGF-β) was up-regulated in Montelukast and MSCs-treated groups with superior effect to MSCs, which explained their underlying mechanism. In addition, the adipose tissue-derived MSCs-treated group had more restoring effects on nasal mucosa structure demonstrated by electron microscopical examination.

Breast cancer is the leading cause of cancer-related deaths in the United States. The majority of deaths (90%) in breast cancer patients is caused by invasion and metastasis–two features related to the epithelial-to-mesenchymal transition (EMT). Twist1 is a key transcription factor that promotes the EMT, which leads to cell migration, invasion, cancer metastasis, and therapeutic resistance. Harmine is a beta-carboline alkaloid found in a variety of plants and was recently shown to be able to induce degradation of Twist Family BHLH Transcription Factor 1 (Twist1) in non-small cell lung cancer cells (NSCLC). In this study, we show that harmine can inhibit migration and invasion of both human and mouse breast cancer cells in a dose-dependent manner. Further study shows that this inhibition is most likely achieved by inducing a proteasome-dependent Twist1 degradation. At the concentrations tested, harmine did not affect the viability of cells significantly, suggesting that its inhibition of cancer cell migration and invasion is largely independent of its cytotoxicity, but due to its ability to affect regulators of EMT such as Twist1. This result may facilitate the development of strategies that target Twist1 to treat metastatic breast cancer, as Twist1 is expressed at a high level in metastatic breast cancer cells but not in normal cells.